Systems, methods, and apparatuses for optical systems in flow cytometers

ABSTRACT

The present set of embodiments relate to a system, method, and apparatus for an optical configuration in a flow cytometer that allows for independent adjustment of focusing for each light source. Such systems, methods, and apparatuses require a final focusing element to be moved near the beginning of the optical train and for each optical element coming after the final focusing element to be configured to accommodate converging light beams while minimizing the introduction of aberrations into those beams.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/633,613 filed Jun. 26, 2017, now U.S. Pat. No. 10,018,553, which is acontinuation of U.S. application Ser. No. 14/607,677 filed Jan. 28,2015, now U.S. Pat. No. 9,726,593, which claims priority to U.S.application No. 62/041,806 filed Aug. 26, 2014, and U.S. application No.61/946,579 filed Feb. 28, 2014, which disclosures are hereinincorporated by reference in their entirety.

FIELD

The present disclosure generally relates to optical systems in flowcytometers.

BACKGROUND

Flow cytometry is a powerful tool used for analysis of particles andcells in a myriad of applications primarily in bioscience research andmedicine. The analytical strength of the technique is in its ability toparade single particles (including bioparticles such as cells, bacteriaand viruses) through a focused spot or spots of light, typically from alaser or lasers, in rapid succession, at rates up to thousands ofparticles per second. The high photon flux at this focal spot producesscatter of light by a particle and or emission of light from theparticle or labels attached to the particle that can be collected andanalyzed. This gives the user a wealth of information about individualparticles that can be quickly parleyed into statistical informationabout populations of particles or cells.

In flow cytometry, multi-beam, multi-wavelength excitation is commonlyused to increase the available number of fluorophores that can act asoptical reporters. The increased spectral space allows for a greaterdegree of assay multi-plexing for individual targets.

Multi-beam flow cytometry can be implemented in several different ways.The simplest is to co-locate beams along the same optical axis. In thissituation, multi-plexing is limited by the spectral overlap offluorophores excited by the wavelength of the co-located beams. In mostsystems, beams are delivered to the flow cell in a stacked manner with asmall distance between beams. This allows for spatially separatedinterrogation zones for each laser or other type of light. In thesesystems, the magnitude of the spatial separation is chosen to reducecross talk between adjacent lasers while minimizing the uncertainty ofparticle position due to fluctuations of the fluidic system. As spatialseparation increases cross talk decreases, but uncertainty of a particleposition increases.

Due to system requirements that mandate high particle analysis rates andhigh illumination intensity, a small laser spot size is desired at atarget. In most systems, a converging beam is required to achieve thislevel of focus on target. A converging beam is typically produced byexpanding laser light (e.g., collimating or partially collimating) andthen propagating it through a focusing lens. For systems with multiplelasers, the collimated optical beams may be stacked with a displacementof a few hundred microns to produce a final spot separation in thatorder. The collimated beams are then passed through a single focusinglens, as seen in FIG. 1, and then propagated to the interrogation zonewhere the particles pass.

The single lens approach has proven effective and has been a mainstayfor many decades. Its drawback is that the focusing lens is coupled toall the beams in the system simultaneously. Adjustment of the singlefocusing lens or implementing other lens manipulations to improve thefocus of one beam degrades the focus of adjacent beams. Such a systemcan produce lower quality data, necessitate more effort in calibratingthe focus, and also makes interchangeability of the light sources (e.g.lasers) extremely difficult.

The solution to these drawbacks, as presented herein, is a system inwhich converging beams can be propagated through the optical traininstead of collimated beams without the introduction of aberrations. SeeFIG. 2. Such a system will allow for adjustment of each laser beamwithout interfering with the optical path of adjacent lasers beams andallow for improved data and results.

SUMMARY

In one aspect, an optical system for a flow cytometer is disclosed. Theoptical system can include a flow cell including a particleinterrogation zone. The optical system can include at least two opticalsubunits comprising a light source producing a light beam and aconverging element configured to convert the light beam into aconverging light beam.

In one aspect, a method to combine light beams in a flow cytometer isdisclosed. The method can include providing at least two light beams.The method can include passing each of the light beams through aconverging element in a one light beam per converging element ratiowherein the light beams leaving the converging elements are converginglight beams. The method can include passing at least one of theconverging light beams through at least one dichroic element. The methodcan include spatially separating the converging light beams from oneanother upon entering the interrogation zone within a flow cell.

In one aspect, a flow cytometer optical alignment method is disclosed.The method can include producing at least two converging light beamswherein each of the converging light beams is produced by passing alight beam produced by a light source through a converging elementwherein the light source and the converging element are affixed to anopto-mechanical mount. The method can include passing at least one ofthe converging light beams through a dichroic element. The method caninclude passing each of the converging light beams through a flow cellin a set of spatially distinct first positions. The method can includeadjusting at least one of the opto-mechanical mounts to reposition atleast one light source and at least one converging element. The methodcan include passing each of the converging light beams through a flowcell in a set of spatially distinct second positions.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

For a more complete understanding of the principles disclosed herein,and the advantages thereof, reference is now made to the followingdescriptions taken in conjunction with the accompanying drawings, inwhich:

FIG. 1 is an illustration of an optical system for a flow cytometeraccording to the prior art.

FIG. 2 is an illustration of an optical system for a flow cytometeraccording to one of the various embodiments.

FIG. 3 is an illustration of a close up view of a portion of an opticalsystem for a flow cytometer according to one of the various embodiments.

FIG. 4 illustrates a flow diagram according to one of the variousembodiments.

FIG. 5A is an illustration of an optical system for a flow cytometeraccording to the prior art using collimated beams.

FIG. 5B is an illustration of an optical system for a flow cytometeraccording to one of the various embodiments using converging beams.

FIG. 6A is a profile of flat top converging laser beams when zero flatplate dichroics are utilized.

FIG. 6B is a profile of flat top converging laser beams when one flatplate dichroics are utilized.

FIG. 6C is a profile of flat top converging laser beams when two flatplate dichroics are utilized.

FIG. 6D is a profile of flat top converging laser beams when three flatplate dichroics are utilized.

FIG. 7A is a profile of flat top converging laser beams when zero cubedichroics are utilized.

FIG. 7B is a profile of flat top converging laser beams when one cubedichroic are utilized.

FIG. 7C is a profile of flat top converging laser beams when two cubedichroics are utilized.

FIG. 8A depicts a blue laser system at focus with associated profiles.

FIG. 8B depicts a violet laser system at focus with associated profiles.

FIG. 8C depicts a blue and violet laser system at focus with associatedprofiles.

FIG. 9A depicts a blue laser system at focus with associated profiles.

FIG. 9B depicts a blue laser system, with a glass window at 45 degrees,at Gaussian focus with associated profiles.

FIG. 9C depicts a blue laser system, with a glass window at 45 degrees,at flat top focus with associated profiles.

FIG. 9D depicts a blue laser system, with two dichroic cubes, at focuswith associated profiles.

FIG. 10A is a plot of intensity and coefficient of variation data for alaser beam focused with no dichroic optical elements in the opticalpath.

FIG. 10B is a plot of intensity and coefficient of variation data for alaser beam propagated through a dichroic cube.

FIG. 10C is a plot of intensity and coefficient of variation data for alaser beam propagated through a dichroic plate at a 45 degree incidentangle.

While the present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications, and equivalents, as will beappreciated by those of skill in the art.

Furthermore, in describing various embodiments, the specification mayhave presented a method and/or process as a particular sequence ofsteps. However, to the extent that the method or process does not relyon the particular order of steps set forth herein, the method or processshould not be limited to the particular sequence of steps described. Asone of ordinary skill in the art would appreciate, other sequences ofsteps may be possible. Therefore, the particular order of the steps setforth in the specification should not be construed as limitations on theclaims. In addition, the claims directed to the method and/or processshould not be limited to the performance of their steps in the orderwritten, and one skilled in the art can readily appreciate that thesequences may be varied and still remain within the spirit and scope ofthe various embodiments.

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiments of systems, methods, and apparatuses for optical systems ina flow cytometer are described in the accompanying description andfigures, which includes Exhibit 1. In the figures, numerous specificdetails are set forth to provide a thorough understanding of certainembodiments. A skilled artisan will be able to appreciate that theoptical system described herein can be used in a variety of instrumentsusing optical trains including, but not limited to, flow cytometers.Additionally, the skilled artisan will appreciate that certainembodiments may be practiced without these specific details.Furthermore, one skilled in the art can readily appreciate that thespecific sequences in which methods are presented and performed areillustrative and it is contemplated that the sequences can be varied andstill remain within the spirit and scope of certain embodiments.

While the present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications, and equivalents, as will beappreciated by those of skill in the art.

Furthermore, in describing various embodiments, the specification mayhave presented a method and/or process as a particular sequence ofsteps. However, to the extent that the method or process does not relyon the particular order of steps set forth herein, the method or processshould not be limited to the particular sequence of steps described. Asone of ordinary skill in the art would appreciate, other sequences ofsteps may be possible. Therefore, the particular order of the steps setforth in the specification should not be construed as limitations on theclaims. In addition, the claims directed to the method and/or processshould not be limited to the performance of their steps in the orderwritten, and one skilled in the art can readily appreciate that thesequences may be varied and still remain within the spirit and scope ofthe various embodiments.

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein “about” means plus or minus 20%, more preferably plus orminus 10%, even more preferably plus or minus 5%, most preferably plusor minus 2%.

As used herein “dichroic” means a wavelength selectively reflectiveelement.

As used herein “flow cell” means a channel, chamber or capillary havingan interior shape selected from rectangular, square, elliptical, oblatecircular, round, octagonal, heptagonal, hexagonal, pentagonal, andtriagonal.

As used herein “channel” means a course, pathway, or conduit with atleast an inlet and preferably an outlet that can contain an amount offluid having an interior shape selected from rectangular, square,elliptical, oblate circular, round, octagonal, heptagonal, hexagonal,pentagonal, and triagonal.

As used herein “particle” means a small unit of matter, to include butnot limited to: biological cells, such as, eukaryotic and prokaryoticcells, archaea, bacteria, mold, plant cells, yeast, protozoa, ameba,protists, animal cells; cell organelles; organic/inorganic elements ormolecules; microspheres; and droplets of immiscible fluid such as oil inwater.

As used herein “analyte” means a substance or material to be analyzed.

As used herein “probe” means a substance that is labeled or otherwisemarked and used to detect or identify another substance in a fluid orsample.

As used herein “target” means anything coming into contact with theconverging beam.

As used herein “interrogation zone” is the point where the light beam(e.g. a laser) intersects with the particle or the place where theoptics system detects light scatter and fluorescence.

As used herein “final focusing lens” means a converging element that islocated somewhere in the optical train.

As used herein “fluid stream” means the stream which carries and alignsthe particles so that they pass single file through the light beam.

In various embodiments, the optical system disclosed in the presentapplication can be used in conjunction with various apparatuses,systems, and methods relating to flow cytometry. See U.S. patentapplication Ser. Nos. 12/239,390 and 12/209,084, both of which areincorporated by reference in their entirety.

In flow cytometry, multi-beam excitation is common to increase theavailable number of fluorophores that can be used as optical reporters.The increased spectral space allows for a greater degree of assaymulti-plexing for individual targets. Multi-beam flow cytometry can beimplemented in several different ways. The simplest is to co-locatebeams along the same optical axis. However, someone skilled in the artwill appreciate that interference, spectral overlap and cross-talk willlimit the degree of multi-plexing. To reduce this problem beams aredelivered to the flow cell in a stacked manner.

Referring to FIG. 1, a schematic of a prior art optical system in thefield of flow cytometry is shown. This system is comprised of at leastone light source 102 that produces a collimated light beam 104. Priorart systems use plate dichroic elements 106 through which the collimatedlight beams 104 pass through. The collimated light beam 104 stayscollimated as it passes through the dichroic elements 106 in order to befocused at the end of the optical train. One effect of collimation isthat it minimizes the introduction of aberrations as the light beampasses through plate dichroic elements 106. If converging beams wereused such aberrations would result in poor data quality and are asubstantial concern in optical systems such as the ones used in flowcytometry. When the light beam hits a target or particle within theinterrogation zone the beam profile has an area commonly referred to as“spot size.” Generally, if a light beam can be fit into a smaller spaceits beam intensity increases which results in higher signal emissionrelative to the background noise. In order for the spot size of thelaser to have a high signal to background noise ratio a convergingelement 108 is used at the end of the optical train to reduce the spotsize of the beam and increase the beam intensity prior to interrogationof a target. As is the case in almost all optical systems, reductions inaberrations and proper focusing are desirable. It is common in flowcytometry to spatially separate (stack) light beams so that a target canbe interrogated by different light sources 102 at different timeswithout light sources 102 interfering with one another or creatingissues involving cross-talk. The biggest problem with the system asshown in FIG. 1 is several collimated light beams 104 are entering thecollimated converging element 108 in a stacked manner and in order toproperly focus an individual light beam the converging element 108 mayneed to be moved. The beams are coupled through the converging element108 and movement of the lens to adjust one beam could negatively impactthe adjustment of adjacent light beams. Additionally, properlycalibrating such an optical system can involve several complex lensmanipulations and despite best efforts may still result in less thanperfect calibration of the optical system. The quality of the resultingdata can be poor which can lead to important information not beingdetectable above the background noise and result in missed data in basicresearch or improper diagnoses in medical applications.

One embodiment of a solution to the problem presented in FIG. 1 isillustrated in FIGS. 2, 3, and 5B where the optical path of each lightsource 102 can be made independent of the other light sources 102 bymoving the final focusing element to the front of the optical path. Thisdistinction can be seen when comparing the location of the convergingelements 108 in FIG. 1 and FIG. 5B. Such a system can have oneconverging element 108 paired with one light source 102. This systemmakes optical alignment in manufacturing and in the field much simplerby decoupling many of the lens manipulations conducted within eachindividual light source path. A modular method whereby fully assembledoptical subunit 502 with all the optical hardware necessary to produce aconverging light beam 206 that focuses to the final spot size can beinstalled into an instrument or removed and replaced. Alignment of theoptical system is then a simple manipulation of the optical subunit 502,whereby, only one optical path can be adjusted at a time. The standardmethod, FIG. 1, for combining the converging light beams 206 in astacked or collinear manner would utilize plate dichroic elements 106 todirect the converging light beams 206. But, this would not be possiblefor the optical setup described above that produces converging lightbeams 206 at the beginning of the optical path. Aberrations (astigmatismand coma) would be introduced by the plate dichroic elements 106 therebydegrading the final spatial distribution of the laser intensity profileand thereby degrading the performance of the instrument. The aberrationsintroduced into converging light beams 206 by plate dichroic elements106 are described in the known art. J. Bratt, Analytical expressions forthe wave front aberration coefficients of a tilted plane-parallel plate,Applied Optics 36, 8459-8467 (1997). J. van den Eerenbeemd and S.Stallinga, Compact system description for systems comprising a tiltedplane parallel plate, Applied Optics 46, 319-326 (2007).

As shown in FIG. 1, an optical system for a flow cytometer can include aflow cell 110 and at least two optical subunits 502 as seen in FIG. 5B.The flow cell 110 can include a particle interrogation zone. In general,a light beam can be focused onto an interrogation zone wherein particlescan pass through the focused light beam. An optical subunit 502 cancomprise a light source 102 and a converging element 108. In variousembodiments the light source 102 can produce a substantiallymonochromatic light beam. In various embodiments the light source 102can produce polychromatic light beams. The converging element 108 can beconfigured to convert either monochromatic or polychromatic light into aconverging light beam 206. In various embodiments, the converging lightbeam can focus to a diameter of about ten micrometers. In variousembodiments, the converging light beam can focus to a diameter ofbetween about one micrometer and twenty micrometers. In variousembodiments, the converging light beam can focus to a diameter ofbetween about fives micrometers and one hundred micrometers. In variousembodiments the light beam entering the converging element 108 caneither be expanding, converging, or collimated. In various embodimentsthe optical subunits 502 can be affixed to an opto-mechanical mount. Theopto-mechanical mounts can be manipulated by an operator to adjust thespatial separation of the converging light beams 206 or can bemanipulated to adjust the focus of the converging light beams 206 withina flow cell 110. Such a system will allow for adjustment of each laserbeam without interfering with the optical path of adjacent lasers beamsand allow for improved data and results.

In various embodiments, the optical system further comprises at leastone dichroic element 204 that is configured to direct the converginglight beams 206 to the flow cell 110. The dichroic element 204 can becomprised of two adjoined prisms 304 and a wavelength selective coating302 located between the adjoined prisms 304. In various embodiments, anelement can be used that is trichroic or more and can make use of anynumber of prisms 304 and any number of wavelength selective coatings302. In various embodiments, the dichroic element 204 used can be in theshape of a cube. A cube shape can allow for entry faces to be nearperpendicular to the incoming beams and the exit faces can be nearperpendicular to the transmitted converging light beams 206. In variousembodiments, the cube structure can reduce or almost eliminate theintroduction of aberrations. In various embodiments, the cube can beconstructed by joining two 45 degree prisms 304. In various embodiments,a wavelength selective coating 302 can be placed on the adjoiningsurface between the prisms 304. Because the propagation of a convergingwave front through a cube allows for fewer and less pronouncedaberrations in the converging light beam 206, it is not necessary topropagate collimated light beams 104 through the dichroic cube. Invarious embodiments, the final focusing lens (e.g. the convergingelement 108 in FIG. 5B) in a multi-laser system can be relocated earlierin the optical path where it can be decoupled from the other opticalaxes. In such embodiments, the lens assemblies with independentconverging elements 108 can be mounted on a three axis opto-mechanicalmount with independent mechanical adjustments for aiming and focusingthe converging light beam 206. In various embodiments, the location ofeach beam waist and position can be individually adjusted for eachconverging light beam 206 without affecting the focus of the otherconverging light beams 206. Such configurations also eliminate issuescaused by chromatic and spherical aberrations that are compounded whenseveral beams of different wavelengths are propagated through the samecollimated converging element 108 and having the requirement to focus inthe same plane. In the various embodiments using dichroic cubes (orother geometries with similar properties), a converging light beam 206configuration of multiple light sources 102 can be constructed where theadjustment of the optical components of each light beam optical path aredecoupled. Additionally, such embodiments allow for the creation amodular flow cytometer. In the various embodiments that decouple thelight beam lines, adding additional light beams lines does not requirehaving to adjust coupled optical components. Such embodiments areespecially advantageous in the field in and manufacturing environments.

The combination of at least two optical subunits 502 and at least onedichroic element 204 has not been practiced before. One reason could bethe increased cost of dichroic elements 204 in the form of cubes orother geometries versus plate dichroic elements 204 which contain muchless material. Additionally, optical subunits 502 are more complicatedto produce.

In various embodiments the wavelength selective coating 302 acts as along pass filter. In other embodiments the wavelength selective coating302 acts as a short pass filter. In additional embodiments, mirrorelements 112 can be used in the various embodiments of the presentoptical system.

In various embodiments, the opto-mechanical mounts can be adjusted on anx, y, and z coordinate system. Placing the optical subunits 502 onopto-mechanical mounts 503 can allow adjustment of each light source 102independent of other light sources 102 being used to create the properfocus and spot size with fewer and less pronounced aberrations requiredfor the particular application. Plate dichroic elements 106 have beenused in the past because they work very well for the majority ofapplications at a reduced cost. However, the various embodiments of thepresent system are superior to that of a plate dichroic systemtransmitting collimated light beams 104 for the above described reasons.

In various embodiments, a particle moves through the flow cell 110. Insuch embodiments, the particle can be interrogated by each of theconverging light beams 206 independently.

In various embodiments, the converging element 108 can be a convex lens.

In various embodiments, the spatial separation can be between about 80micrometers to about 200 micrometers when passing through theinterrogation zone. In various embodiments, the spatial separation canbe between about 10 micrometers to about 100 micrometers. In variousembodiments, the spatial separation can be about 150 micrometers.

In various embodiments, the converging light beam 206 can have a flattop intensity profile. In various embodiments, the converging light beam206 can have a Gaussian intensity profile. In various embodiments, theintensity profile can be altered depending on the application.

FIG. 4 is an exemplary flowchart showing a method 400 for flow cytometeroptical alignment, in accordance with various embodiments.

In step 402, at least two light beams can be provided. In step 404, eachof the light beams can be passed through a converging element in a onelight beam per converging element ratio wherein the light beams leavingthe converging elements can be converging light beams 206. In step 406,at least one of the converging light beams 206 can be passed through atleast one dichroic element 204. In step 408, the converging light beams206 can be spatially separated from one another upon entering theinterrogation zone within a flow cell 110.

In various embodiments of the method, the light beams can bemonochromatic light emissions and can be produced by the light source102. In various embodiments, the light source 102 and the convergingelement 108 can be affixed to an opto-mechanical mount. In variousembodiments, the opto-mechanical mount can be adjusted on an x, y, and zcoordinate system. In various embodiments, a further step can includereplacing a first light source 102 configured to produce a monochromaticlight emission with a first wavelength with a second light source 102configured to produce a monochromatic light emission with a secondwavelength.

In various embodiments of the method, the dichroic element 204 can beconfigured to prevent introduction of aberrations into the converginglight beams 206. In various embodiments, the dichroic element 204 can bein the shape of a cube. In various embodiments, the dichroic elements204 can be long pass filters or short pass filters. In variousembodiments, a further step can include replacing at least one firstdichroic element 204 configured to pass a first range of wavelengthswith at least one second dichroic element 204 configured to pass asecond range of wavelengths.

In various embodiments of the method, the adjustment of theopto-mechanical mounts can occur on an x, y, and z axis.

In various embodiments of the method, a further step can includeinterrogating a particle wherein the particle is passing through theflow cell 110 in a fluid stream. In various embodiments, the fluid canbe a liquid. In various embodiments, a further step can includedetecting light scatter with a detection element. In variousembodiments, the detection element can comprise a photo multiplier tube.In various embodiments there can be forward and side scattered lightgenerated when the converging light beam 206 strikes the target. Invarious embodiments, side scattered light can pass through a detectionlens and then enter a pinhole collection fiber array wherein eachpinhole can correspond to a specific converging light beam 206 that hasentered the flow cell 110 in a spatially separated manner. In variousembodiments the spatial separations can range from about 80 micrometersto about 200 micrometers. In various embodiments, the spatial separationcan be between about 10 micrometers to about 100 micrometers. In variousembodiments, the spatial separation can be about 150 micrometers. Invarious embodiments, fiber optic cables can connect the light coming outof each pinhole to a collection block. In various embodiments acollection block can include collimators, filter elements, and photomultiplier tubes. In various embodiments the signal can be convertedfrom analog to digital data which can then be stored and analyzed on acomputer. In various embodiments, forward scattered light can pass ablocker bar, condenser lens, and be converted from analog to digitaldata signal. In various embodiments, the digital data signal can then bestored and analyzed on a computer.

In various embodiments, a further step can include applying hydrodynamicand or acoustic focusing to the particle.

In various system, apparatus, and method embodiments, an optical subunit502, a dichroic element 204, and collection block can be associated witha first wavelength of light. In various embodiments, the optical subunit502, the dichroic element 204, and the collection block described abovecan be removed from the rest of the flow cytometer system and thenreplaced with a different optical subunit 502, dichroic element 204, andcollection block that are associated with a second wavelength of light.In various embodiments, the computer where the digital data is storedcan be programmed with this change in optical components and thenanalyze data using a new set of parameters.

In various embodiments of the method, the dichroic element 204 can beconfigured to prevent introduction of aberrations into the converginglight beams 206. As previous discussed, plate dichroic elements 106, asseen in FIG. 3, introduce aberrations into converging light beams 206,but not collimated light beams 104. In various embodiments, the dichroicelement 204 can be in the form of a cube which can allow all parts ofthe converging light beams 206 to enter the cube at the same time whichthen allows the converging light beams 206 to enter a new medium with adifferent index of refraction with little or no introduction ofaberrations. In this configuration, the exiting converging light beams206 can enter air or another medium from which they originated withoutthe introduction of aberrations on the back end as well.

In various embodiments of the method, the converging light beams 206 canhave a flat top intensity profile. Such a flat top intensity profile canallow for uniform interrogation of a target whether that target is aparticle, cell, bead, or other.

Now referring to FIG. 5A and FIG. 5B, a further comparison of prior artand the present disclosure are illustrated. In FIG. 5A light sources 102can be seen producing collimated light beams 104. After some light beammanipulations occur (not shown) the collimated light beams 104 passthrough a converging element 108 and then strike a target 504. In thisillustration there are three light beams all passing through a singleconverging element 108. Each light beam must be focused by moving theconverging element 108. It is apparent in this illustration thatfocusing one light beam by changing the position of the convergingelement 108 will impact the other two light beams because all threelight beams pass through the same converging element 108. Such a systemis inferior to the system seen in FIG. 5B.

FIG. 5B depicts one example embodiment of an optical system for a flowcytometer. In FIG. 5B, each converging light beam 206 can be producedfrom an optical subunit 502 containing both a light source 102 and aconverging element 108. In such a system, an optical subunit 502 can bemounted to an opto-mechanical mount (not shown) to focus and adjust aconverging light beam 206. Such a system can allow for adjustment,commonly focus or spot size, for each converging light beam 206independent of other converging light beams 206. Such a system provideshigher data quality, easier configuration, and modularity as detailedthroughout this specification.

The following examples are offered to illustrate, but not limit, theembodiments disclosed herein.

Example 1 Flat-Top, Converging Laser Beam Profile Changes When PassingThrough Flat Plate Dichroics Set at a 45 Degree Angle

Converging laser beams were passed through between 0 and 3 flat platedichroics (FIGS. 6A-6D) and the resulting beams were imaged by a camera.The x-axis represents beam position and the y-axis represents beamintensity. Intensity on the y-axis has been normalized to 1 (or 100%)where 1 is the highest intensity reached. For most applications, thebeam width should be about 50 micrometers across at the 90%. In variousembodiments, the beam width can be about 40 micrometers across or about10 micrometers across while maintaining a beam intensity of at leastabout 90%. In various embodiments, the beam intensity should be at leastabout 80%, at least about 70%, at least about 60%, or at least about50%, while maintaining a beam width of at least about 50 micrometersacross. The optimal beam can be seen when 0 dichroic plates (FIG. 6A)are used, but when even 1 flat plate dichroic (FIG. 6B) is addedconditions for optimum interrogation of a particle are not met.

This example illustrates that combining the converging light beams 206technology with plate dichroic elements 106 technology is not optimal.As discussed above, converging light beams 206 allow for the productionof independent optical trains. Therefore, another technology (e.g.dichroic elements in the form of prisms) had to be incorporated intothese various embodiments.

Example 2 Flat-Top, Converging Laser Beam Profile Changes When PassingThrough Cube Shaped Dichroics

Converging laser beams were passed through between 0 and 2 cubedichroics (FIGS. 7A-7C) and the resulting beams were imaged by a camera.The x-axis represents beam position and the y-axis represents beamintensity. Intensity on the y-axis has been normalized to 1 (or 100%)where 1 is the highest intensity reached. For optimum interrogationconditions the beam width should be about 50 micrometers across and atleast about 90% intensity level or higher. 50 micrometers was chosenbecause the area allows for a high enough beam intensity to be effectivewhile maximizing spot size. The optimal beam can be seen when 0, 1, or 2dichroic cubes are used.

This example demonstrates a dichroic solution that is compatible withconverging light beams 206. Unlike Example 1, there is almost nodegradation in the beam profile which can allow for the production ofindependent optical paths.

Example 3 Wavefront Aberration for a Dichroic Plate Set at a 45 DegreeAngle

Coefficient Value (1/π) Aberration W40 0.0431 Lowest Order SphericalAberration W60 0.0004 Sixth Order Spherical Aberration W22 2.2018 LowestOrder Spherical Aberration W42 −0.0204 Fifth Order Astigmatism W31−0.7685 Third Order Linear Coma W51 −0.0065 Fifth Order Linear Coma W33−0.0443 Cubic Coma

T=1 mm; n=1.5145; λ=637 nm; pupil radius=9 mm; tilt=45 deg; r=1

${\Delta\; W} = {\sum\limits_{l}{\sum\limits_{m}{W_{lm}\rho^{l}\cos\; m\;\varphi}}}$

Example 3 presents well known calculations demonstrating why usingdichroic plates in a converging light (e.g. laser) system produceunacceptable beam profiles with significant astigmatism and comaaberrations. Such aberrations reduce the quality of data substantially.T=thickness of the plate used; n=index of refraction; λ=wavelength oflight; pupil radius=spot size on glass where calculation is occurring;tilt=angel of plate; and r=distance from the optical axis (exit of pupilradius).

Example 4 Wavefront Aberration for a Dichroic Cube

Coefficient Value (1/π) Aberration W40 0.6345 Lowest Order SphericalAberration W60 0.0094 Sixth Order Spherical Aberration W22 0.000 LowestOrder Spherical Aberration W42 0.000 Fifth Order Astigmatism W31 0.000Third Order Linear Coma W51 0.000 Fifth Order Linear Coma W33 0.000Cubic Coma

T=20 mm; n=1.5145; l=637 nm; pupil radius=9 mm; tilt=0 deg; r=1

See Above for Equation

Example 4 presents well known calculations that demonstrate that usingdichroic cubes in place of dichroic plates results in no coma orastigmatism. These reductions lead to much higher quality data ascompared to an instrument that incorporates converging light (e.g.laser) beams into a dichroic plate optics system.

Example 5 Co-Plannar Focus

The data in Example 5 presents the use of two optical subunits eachcomprising a light source and a converging element being focusedindependently without the use of a final focusing lens. The subunits inthis example include an opto-mechanical mount that can be spatiallyadjusted. More specifically, the light sources in the above example area blue laser and a violet laser. The focusing occurred through use ofthe optical subunits which is discussed in greater detail throughout thecurrent disclosure.

FIG. 8A, entitled “Blue Laser Beam at Focus”, is a two laser systemwhere a blue laser beam has been focused into a camera independently ofa violet laser beam which has been blocked with a blocking device. Foroptimal interrogation conditions within a flow cytometer the beam widthis about 50 micrometers across at least about 90% intensity level orhigher. The Gaussian focus is about 2 micrometers. Laser beam profileand focusing can have different optimal configurations depending on theapplication.

FIG. 8B, entitled “Violet Laser Beam at Focus”, is the same two lasersystem where a violet laser beam has been focused into a cameraindependently of a blue laser beam which has been blocked with ablocking device. For optimal interrogation conditions within a flowcytometer the beam width is about 50 micrometers across at least about90% intensity level or higher. The Gaussian focus is about 2micrometers. Laser beam profile and focusing can have different optimalconfigurations depending on the application.

FIG. 8C, entitled “Blue and Violet Laser Beams at Focus”, presents datademonstrating that both laser beams and their beam shaping optics havebeen focused independently using optical subunits which includes anopto-mechanical mount system. The lower left portion showing the flattop laser beam profiles has been normalized to the blue laser beam whichis why the violet laser beam shows a beam intensity that doesn't spanthe height of the graph. However, it is shown that about 90% of eachlaser's intensity occurs over a beam width of about 50 micrometers aftereach subunit has been adjusted independently. The Gaussian focus foreach laser beam is about 2 micrometers. Such adjustment using a finalfocusing lens, as used in the prior art, would not allow for independentadjustment to focus each laser beam. The detractor of a final focusinglens is that while optimizing one laser beam profile the user may bereducing the optimization of another laser beam profile. Such a problemdoes not occur in a system where independent beam profile optimizationis possible.

Example 6 Reduced Aberration Beam Stacking

Example 6 compares the amount and severity of aberration introduced intoan optical system when a laser beam is passed through a 45 degree flatwindow (e.g. a dichroic plate) with a width of 3 mm versus a dichroiccube element, two dichroic cubes, or through no optical elements.

FIG. 9A, entitled “Blue Laser Beam at Focus”, shows a blue laser beamfocused into a camera without the use of any dichroic elements. Datafrom a flat top beam is presented in the lower left graph with about 90%of the beam intensity covering a flat top width of about 47 micrometers.The focus is at about 2 micrometers on the Gaussian axis.

FIG. 9B, entitled “Blue Laser Beam with a 3 mm 45 Degree Window—AtGaussian Focus”, presents data from a blue laser beam focused at acamera with a glass window titled at about 45 degrees incident to thelaser beam between the laser source and camera. The glass window isabout 3 millimeters thick and constructed from glass. The focus is atabout 2 micrometers on the Gaussian axis. The lower left graph showsthat there is no longer a flat top beam profile and that only about 2.5micrometers of the beam width goes above about 90% beam intensity. Thedifference between this data and the data from the first figure is dueto introduction of aberrations from passing the laser beam through theoptical element. Such an optical configuration is not suitable for avariety of instruments requiring specific optical conditions, includingflow cytometry. When a beam profile takes on the characteristic shown inthis figure an abundance of noise is created within the data.

FIG. 9C, entitled “Blue Laser Beam with a 3 mm 45 Degree Window—At FlatTop Focus”, presents data from a blue laser beam focused at a camerawith a glass window titled at about 45 degrees incident to the laserbeam. The glass window is about 3 millimeters thick and constructed fromglass. The focusing was determined by where the maximum flat top profileintensity and width occurred. The reverse is occurring in this figure asthe last. In this case, the flat top beam with is at about 90% intensityfor about 48 micrometers. However, the Gaussian has gone well over about2 micrometers at about 90% beam intensity (to about 13.7 micrometers).The difference between this data and the data from the first figure isdue to introduction of aberrations from passing the laser beam throughthe optical element. Noise in the acquired data will also increase withthis beam profile.

FIG. 9D, entitled “Blue Laser Beam at Focus with 2 Dichroic Cubes”,presents data from a blue laser being focused through two opticalelements (dichroic cubes) and onto a camera. When compared to the firstfigure in this example, the laser beam profile matches very closelywhich shows that very little, if any, aberrations have been introducedby the two dichroic cubes. This is because the flat top laser beamprofile is maintained when propagated through two dichroic cubes. Theflat top focus has a beam width of about 51.9 micrometers and theGaussian focus has a width of about 2 micrometers. Such a beam profilewill produce high quality data unlike the beam profile coming out of theabout 3 millimeter glass windows which will likely produce unusable datain flow cytometry and other applications.

Example 7 Percent Coefficient of Variation

In flow cytometry the degree to which replicate measurements of aparticle agree with one another can be characterized by the coefficientof variation (CV). A measure of the variability in signal intensity isgenerated as particles pass repeatedly though a light source (e.g. alaser beam). The variability is expressed as a percentage of the averagesignal intensity. This statistical measurement is well known in the artand is defined as 100 times the standard deviation divided by the mean.Generally, a lower CV means that replicate measurements agree with oneanother. Each of the following figures present two graphical plots. Thedotted plots display CV and the dashed plots display intensity. They-axis is normalized to percentage where 1.0 is 100 percent. The x-axishas units in micrometers. The laser beam is converging in each of thesedata sets. The data following data was collected using a Data RayBeam'R2. Laser focus was optimized by focusing the beam height to about10 micrometers. The coefficients of variation (CVs) were calculatedusing about a 50 micrometer window width. The plots shown in FIGS.10A-10C display normalized intensity as well as a moving window CV.

The data in FIG. 10A, entitled “Intensity and CV for a Beam in FreeSpace”, was acquired by focusing a laser beam onto a camera with nodichroic optical elements in the optical path. The data shows a flat toplaser beam profile where about 50 micrometers of the laser beam is aboveabout the 90% intensity level. The CV drops to about 0.5 or about 5%.

The data in FIG. 10B, entitled “Intensity and CV for a Beam PropagatedThrough about a 15 mm BK7 Dichroic Cube”, was acquired by focusing alaser beam onto a camera where the laser beam was propagated throughabout a 15 millimeter BK 7 dichroic cube. BK7 glass is known for itshigh rate of transmission and for having an index of refraction of about1.5 depending on the wavelength of light being transmitted. The datashows a flat top laser beam profile where about 50 micrometers of thelaser beam is above about the 90% intensity level. The CV drops to about0.5 or 5%. More specifically, the laser beam profile is unchanged withlittle or no aberrations being introduced.

The data in FIG. 10C, entitled “Intensity and CV for a Beam PropagatedThrough about a 3 mm BK7 Plate at about a 45 Degree Incident Angle”, wasacquired by focusing a laser beam onto a camera where the laser beam waspropagated through about a 3 millimeter BK 7 dichroic plate at about a45 degree angle of incidence. The data shows a laser beam profile whereabout significantly less than about 50 micrometers of the laser beam isabove about the 90% intensity level. The CV is of such poor quality thatthe y-axis has been altered to accommodate the high CV. A laser beamprofile such as this has had multiple severe aberrations introduced.Data collected would contain large amounts of noise and could beunusable.

In this example, it is clearly shown that the standard flat platedichroic plates are unable to accommodate the converging laser beam inthis system. A preferable CV is about 10% or less. A more preferable CVis about 9% or less. An even more preferable CV is about 8% or less. Aneven more preferable CV than 8% is about 7% or less. An even morepreferable CV than 7% is about 6% or less. An even more preferable CVthan 6% is about 5% or less. An even more preferable CV than 5% is about4% or less. An even more preferable CV than 4% is about 3% or less. Aneven more preferable CV than 3% is about 2% or less. A most preferableCV is about 1% or less.

The current optical system can achieve such CV values from eachindividual light source (e.g. laser beam) by being able to independentlyfocus the light source into a converging light beam at the beginning ofthe optical train and then pass that converging light beam throughdichroic cubes instead of the dichroic plate (ubiquitous optical elementin the art) that is often angled at about 45 degrees incident to theincoming light source.

An optimal light beam profile has a flat top. An optimal light beamwidth is between about 30 to about 70 micrometers. A more optimal lightbeam width is between about 35 to about 65 micrometers. A more optimallight beam width is between about 40 to about 60 micrometers. A moreoptimal light beam width is between about 45 to about 55 micrometers.

An optical train can comprise one or more dichroic cubes, one or moredichroic plates, one or more mirrors, one or more lenses, one or morespinning disks, one or more filter wheels, one or more objectives,elements capable of reflection or transmission, or any known opticalelement in the art. Additionally, optical elements can comprise plastic,glass, or any other known or useful material or combination ofmaterials. For example, a glass surface can be coated with a reflectivematerial where the reflective material can be a material other thanglass.

While the present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications, and equivalents, as will beappreciated by those of skill in the art.

Further, in describing various embodiments, the specification may havepresented a method and/or process as a particular sequence of steps.However, to the extent that the method or process does not rely on theparticular order of steps set forth herein, the method or process shouldnot be limited to the particular sequence of steps described. As one ofordinary skill in the art would appreciate, other sequences of steps maybe possible. Therefore, the particular order of the steps set forth inthe specification should not be construed as limitations on the claims.In addition, the claims directed to the method and/or process should notbe limited to the performance of their steps in the order written, andone skilled in the art can readily appreciate that the sequences may bevaried and still remain within the spirit and scope of the variousembodiments.

What is claimed:
 1. A system, comprising: a flow cell configured toreceive a fluid sample, the flow cell having an interrogation zone; afirst optical subunit comprising: (i) a first light source configured toproduce a first light beam, and (ii) a first converging elementconfigured to convert the first light beam into a first converging lightbeam; a second optical subunit comprising: (i) a second light sourceconfigured to produce a second light beam, and (ii) a second convergingelement configured to convert the second light beam into a secondconverging light beam; and an optical train comprising a dichroicelement configured to direct the first and second converging light beamsto the interrogation zone, wherein the first and second converging lightbeams are communicated directly from the dichroic element to theinterrogation zone.
 2. The system of claim 1, wherein the dichroicelement is configured to receive the first converging light beam throughan entry face, and wherein the entry face is configured to be at leastnear perpendicular to the first converging light beam.
 3. The system ofclaim 2, wherein the dichroic element is configured to transmit thefirst converging light beam as a first transmitted converging light beamthrough an exit face, and wherein the exit face is configured to be atleast near perpendicular to the first transmitted converging light beam.4. The system of claim 3, wherein the dichroic element is additionallyconfigured to receive the second converging light beam through a secondentry face, and wherein the second entry face is configured to be atleast near perpendicular to the second converging light beam.
 5. Thesystem of claim 4, wherein the dichroic element is configured totransmit the second converging light beam as a second transmittedconverging light beam through the exit face.
 6. The system of claim 2,wherein the entry face is configured to allow all parts of the firstconverging light beam to simultaneously enter the dichroic element. 7.The system of claim 1, wherein the dichroic element comprises prisms. 8.The system of claim 7, wherein the dichroic element comprises two prismsarranged to form a cube.
 9. The system of claim 8, wherein the dichroicelement includes a wavelength selective coating located between the twoprisms.
 10. The system of claim 1, wherein the optical train isconfigured to maintain a flat top profile or a Gaussian profile of thefirst and second converging light beams as the first and secondconverging light beams are directed to the interrogation zone.
 11. Thesystem of claim 1, wherein the optical train is configured to introducelittle or no aberrations into the first and second converging lightbeams as the first and second converging light beams are directed to theinterrogation zone.
 12. The system of claim 11, wherein the aberrationsare spherical aberrations, astigmatisms, linear comas and cubic comas.13. The system of claim 1, wherein the system further comprises: a thirdoptical subunit comprising: (i) a third light source configured toproduce a third light beam, and (ii) a third converging elementconfigured to convert the third light beam into a third converging lightbeam; and wherein the optical train is configured to direct the thirdconverging light beam to the interrogation zone.
 14. The system of claim1, wherein the optical train is configured to direct the first andsecond converging light beams to the interrogation zone with spatialseparation upon entry of the interrogation zone.
 15. The system of claim14, wherein the first and second optical subunits are each affixed to anopto-mechanical mount, wherein each opto-mechanical mount is operable toadjust the spatial separation of the first and second converging lightbeams.
 16. The system of claim 1, wherein the first and secondconverging elements are convex lenses.
 17. The system of claim 1,wherein the optical train does not include a plate dichroic element. 18.The system of claim 1, wherein the first light beam and the second lightbeam are not collimated.
 19. The system of claim 1, wherein the flowcell is configured to receive a flow with a plurality of particlesthrough the interrogation zone.